1 / 75

Lecture 3 RNA Biosynthesis (Transcription)

Lecture 3 RNA Biosynthesis (Transcription). RNA. Transcription. The synthesis of RNA molecules using DNA strands as the templates so that the genetic information can be transferred from DNA to RNA. Transcription. DNA. Similarity between replication and transcription.

flint
Télécharger la présentation

Lecture 3 RNA Biosynthesis (Transcription)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Lecture 3RNA Biosynthesis (Transcription)

  2. RNA Transcription • The synthesis of RNA molecules using DNA strands as the templates so that the genetic information can be transferred from DNA to RNA. Transcription DNA

  3. Similarity between replication and transcription • Template: both processes use DNA as the template. • Bond: phosphodiester bonds are formed in both cases. • Direction: both synthesis directions are from 5´ to 3´.

  4. Differences between replication and transcription

  5. Section 1 Template and Enzymes

  6. §1.1 Template • The wholegenome of DNA needs to be replicated, but only small portion of genome is transcribed in response to the development requirement, physiological need and environmental changes. • Structural genes: DNA regions that can be transcribed into RNA.

  7. The template strand and the coding strand • The template strand: the strand from which the RNA is actually transcribed. It is also termed as antisense strand. • The coding strand: its sequence is the same as the newly created RNA transcript (except for the substitution of uracil for thymine). It is also called as sense strand. Transcription

  8. Asymmetric transcription • Only the templatestrand is used for the transcription, but the coding strand is not. • Both strands can be used as the templates. • The transcription direction on different strands is opposite. coding strand template strand template strand coding strand

  9.   holoenzyme   Core enzyme  §1.2 RNA Polymerase • The enzyme responsible for the RNA synthesis is DNA-dependent RNA polymerase(DDRP). • The prokaryotic RNA polymerase is a multiple-subunit protein of ~480kD. • The holoenzyme of RNA-pol in E.coli consists of 5 different subunits: 2.

  10. RNA-pol of E. Coli • Rifampicin, a bactericidal antibiotic drug, typically used to treat Mycobacterium infections, including tuberculosis and leprosy. • It can bind specifically to the  subunit of RNA-pol, and inhibit the RNA synthesis.

  11. §1.3 Recognition of Origins • Promoter • The promoter is the DNA sequence that RNA-pol can bind. It is the key point for the transcription control.

  12. Prokaryotic promoter • The -10 region of TATAAT is the region at which a stable complex of DNA and RNA-pol is formed. • The -35 region of TTGACA sequence is the recognition site and the binding site of RNA-pol. Consensus sequence

  13. ß’   ß   DNA UP -35 -10 Prokaryotic promoter TTGACA TATAATstart

  14.  subunit: recognize the promoter. • Holoenzyme bind to the promoter.

  15. Section 2 Transcription Process

  16. General concepts • Three phases: initiation, elongation, and termination. • The prokaryotic RNA-pol can bind to the DNA template directly in the transcription process. • The eukaryotic RNA-pol requires co-factors to bind to the DNA template together in the transcription process.

  17. §2.1 Transcription of Prokaryotes • Initiation: RNA-pol recognizes the promoter and starts the transcription. • Elongation: the RNA strand is continuously growing. • Termination: the RNA-pol stops synthesis and the nascent RNA is separated from the DNA template.

  18. a. Initiation • RNA-pol recognizesthe TTGACA region, and slides to the TATAAT region, then opens the DNA duplex (open complex). • The unwound region is about 171 bp.

  19. The first nucleotide on RNA transcript is always purine triphosphate. GTP is more often than ATP. • The pppGpN-OH structure remains on the RNA transcript until the RNA synthesis is completed. • The three molecules form a transcription initiation complex. RNA-pol (2) - DNA - pppGpN- OH 3

  20. No primer is needed for RNA synthesis. • The  subunit falls off from the RNA-pol once the first 3,5 phosphodiester bond is formed. • The core enzyme moves along the DNA template to enter the elongation phase.

  21. The σcycle • The σfactor dissociates and joins with a new core polymerase to initiate another RNA chain.

  22. Transcription initiation complex RNApol (2) - DNA - pppGpN- OH 3

  23. b. Elongation • The release of the  subunit causes the conformational change of the core enzyme. • The core enzyme slides on the DNA template toward the 3 end. • Free NTPs are added sequentiallyto the 3-OH of the nascent RNA strand.

  24. Transcription bubble: RNA-pol, DNA segment of ~40nt and the nascent RNA form a complex called the transcription bubble. • The 3 segment of the nascent RNA hybridizes with the DNA template, and its 5end extends out the transcription bubble as the synthesis is processing.

  25. RNA-pol of E. Coli

  26. Simultaneous transcriptions and translation

  27. c. Termination • The RNA-pol stops moving on the DNA template. The RNA transcript falls off from the transcription complex. • Two different strategies for transcription termination • -dependent or -independent manner.

  28.  -dependent termination • A protein factor, factor, destabilizes the interaction between the template and the mRNA, releasing the newly synthesized mRNA from the elongation complex. • The factor, a hexamer, is a ATPase and a helicase.

  29. -independent termination • The termination signal is a stretch of 30-40 nucleotides on the RNA transcript, consisting of G-C rich hairpin loop followed by a series of U.

  30. 不依赖r-因子的终止子：含富GC的回文序列和寡聚U序列。不依赖r-因子的终止子：含富GC的回文序列和寡聚U序列。

  31. Stem-loop disruption • The stem-loop structure alters the conformation of RNA-pol, leading to the pause of the RNA-pol moving. • Then the competition of the RNA-RNA hybrid and the DNA-DNA hybrid reduces the DNA-RNA hybridstability, and causes the transcription complex dissociated. • Among all the base pairings, the most unstable one is rU:dA.

  32. Transcription Cycle Promoter sigma factor Terminator

  33. §2.2 Transcription of Eukaryotes • The eukaryotic RNA-pol requires co-factors to bind to the DNA template together in the transcription process. • Eukaryotic systems have three kinds of RNA polymerases, each of which is a multiple-subunit protein and responsible for transcription of different RNAs.

  34. RNA-pol of eukaryotes • hnRNA: heterogeneous nuclear RNA • Amanitin is a specific inhibitor of RNA-pol.

  35. Be careful of this deadly mushroom! Amanita phalloides ,or death cap α-amanitin • α-amanitin is a cyclic peptide of eight amino acids. It is possibly the most deadly of all the amatoxins. • It is an inhibitor of RNA polymerase II. This mechanism makes it a deadly toxin.

  36. Transcription of Eukaryotes a. Initiation • Transcription initiation needs promoter and upstream regulatory regions. • Cis-acting elements:the specific sequences on the DNA template that regulate the transcription of one or more genes.

  37. Cis-acting element -30 • Enhancer: DNA sequences that increase the rate of initiation of transcription by RNA pol II

  38. Transcription factors(TF) • RNA-pol does not bind the promoter directly. • RNA-pol II associates with six transcription factors, TFII A - TFII H. • Trans-acting factors: the proteins that recognize and bind directly or indirectly cis-acting elements and regulate its activity.

  39. TF for eukaryotic transcription

  40. Pre-initiation complex (PIC) • TBP of TFII D binds TATA • TFII A and TFII B bind TFII D • TFII F-RNA-pol complex binds TFII B • TFII F and TFII E open the dsDNA (helicase and ATPase) • TFII H: completion of PIC

  41. Order of binding is: IID + IIB + RNA poly. II + IIF +IIE +IIH • TBP: TATA binding protein.

  42. TAF TBP TF II H Pre-initiation complex (PIC)

  43. b. Elongation • The elongation is similar to that of prokaryotes. • The transcription and translation do not take place simultaneously since they are separated by nuclear membrane.

  44. nucleosome RNA-Pol moving direction RNA-Pol RNA-Pol

  45. c. Termination • The termination sequence is AATAAA. • The termination is closely related to thepost-transcriptional modification.

  46. RNA pol can be selectively inhibited • Rifampicin: a bactericidal antibiotic drug • It can bind specifically to the  subunit of the bacterial RNA-pol. • α-amanitin: a deadly toxin. • It is an inhibitor of RNA polymerase II. • Actinomycin D: commonly used in treatment of a variety of cancers. • it can bind DNA duplexes, and prevent elongation by RNA polymerase in both bacteria and eukaryotes.

  47. Section 3 Post-Transcriptional Modification

More Related